Pores regulate access between ferric-oxy biomineral inside and reductants/chelators outside the ferritin protein nanocage to control iron demineralization rates. The pore helix/loop/ helix motifs that are contributed by three subunits unfold independently of the protein cage, as observed by crystallography, Fe removal rates, and CD spectroscopy. Pore unfolding is induced in wild type ferritin by increased temperature or urea (1-10 mM), a physiological urea range, 0.1 mM guanidine, or mutation of conserved pore amino acids. A peptide selected for ferritin pore binding from a combinatorial, heptapeptide library increased the rate of Fe demineralization 3-fold (p < 0.001), similarly to a mutation that unfolded the pores. Conjugating the peptide to Desferal (desferrioxamine B mesylate), a chelator in therapeutic use, increased the rates to 8-fold (p < 0.001). A second pore binding peptide had the opposite effect and decreased the rate of Fe demineralization 60% (p < 0.001). The peptides could have pharmacological uses and may model regulators of ferritin demineralization rates in vivo or peptide regulators of gated pores in membranes. The results emphasize that small peptides can exploit the structural plasticity of protein pores to modulate function.Pores in ferritin protein cages are an example of pores in molecular or ionic barriers; other examples are pores in membranes. In ferritin, pores control reactions between reductants outside the protein and the ferric mineral inside. As with many gated pores, ferritin pores are formed by ␣-helices in multiple subunits that surround the ion path and are modulated by unfolding (1, 2) (see Fig. 1). Ferritin pores are arranged symmetrically around the protein cage, eight for 24 subunit maxi-ferritins and four for 12 subunit maxi-ferritins, also called Dps proteins (2, 3).Ferritins concentrate Fe for biological use and also detoxify Fe/O 2 or H 2 O 2 in the ferric/oxy biomineral inside the proteins of Archaea, bacteria, and Eukaryota, including higher plants and animals. Fe ions destined to enter the mineral appear to reach the ferroxidase coupling site through the pores for the first step in biomineralization (2, 4 -7). The critical roles of ferritins are illustrated by lethality of deletions in mice, neurological effects of mutations in humans, and pathogen responses to host-released oxidants (3,8,9). In addition, dual genetic regulatory systems with DNA (antioxidant-response elements) enhancers linking ferritin regulation to antioxidant response proteins (10, 11) and mRNA "promoters" (iron-response element) linking ferritin regulation to Fe trafficking proteins emphasize the central role of ferritin in Fe and oxygen metabolism. Finally, the ferritin Fe reactions with O 2 or H 2 O 2 (2, 4 -7) and the presence of ferritin in anaerobic archaea (12) suggest an ancient role for ferritins in the transition to aerobic metabolism.The rates of Fe transport from the ferritin mineral through the nanocage channels and pores to chelators on the outside of the nanocage are initiated by...

Cephalosporins remain one of the most important classes of antibiotics. A useful site for derivatization involves generation of and chemistry at the 3′-hydroxymethyl position. While 3′-acetoxymethyl substituted cephalosporins are readily available, deacetylation to access the free 3′-hydroxymethyl group is problematic when the carboxylic acid is protected as an ester. Herein we report that this important transformation has been efficiently accomplished using Candida antarctica lipase B. Although this transformation is difficult to carry out using chemical methods, the enzymatic deacetylation has been successful on gram scale, when the cephalosporin is protected as either the benzhydryl or t-butyl esters, and on the corresponding sulfoxide and sulfone of the tbutyl ester.Cephalosporin antibiotics have been in use for more than 40 years and are still being employed to fight bacterial infections despite the rising incidence of resistance. 1 Decades of intense research have given rise to an arsenal of synthetic methods that have allowed many analogs to be prepared, including four generations of cephalosporins that have reached clinical use. 2 Although this research has led to many breakthroughs in cephalosporin-specific chemical methodology, accessing precursors for further elaboration is still not straightforward. Numerous derivatives have been made through modification at the 3′-hydroxymethyl group of cephalosporins. However, the deprotection of the common 3′-acetoxy precursor in the presence of the protected cephalosporanic acid, and without lactonization and/or double bond isomerization to give the Δ 2 isomer, has yet to be accomplished in a high yielding and reproducible manner (Scheme 1). Herein we report a selective enzymatic approach to the deprotection of the cephalosporin acetoxy group, using commercially available Candida antartica lipase B (CAL B), on acrylic resin. mmiller1@nd.edu. Supporting Information Available: Experimental methods for compounds 1-3 and 5-9, and all 1 H and 13 C NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org. Commercially available 7-amino cephalosporanic acid (7-ACA) was converted, in reasonable yields, to 3 using a modified literature procedure to introduce the t-butyl ester 4 and standard Schotten-Baumann conditions to install the phenylacetyl group as a simple representative side chain (Scheme 2). Despite considerable effort, the 3′-acetoxy substituent was unable to be removed in acceptable yields and in the absence of double bond isomerization using the following methods (see supporting information for details): saponification, 4 KCN, HCl, 5 TMSI/trifluoroacetate/pH 7.0, 6 or bis(tributyltin) oxide. 7 Based on these findings, enzymatic deacetylation methods were explored. NIH Public AccessEnzymatic deprotection of the cephalosporin acetoxy group has been demonstrated in the literature. 8 Similar to chemical deacetylations, 4,6,8c,d,f,9 the enzymatic deacetylation reactions have only been reported when the cephalosporin co...